Estrogen Regulates Estrogen Receptors and Antioxidant Gene Expression in Mouse Skeletal Muscle Kristen A. Baltgalvis 1 *, Sarah M. Greising 2 , Gordon L. Warren 3 , Dawn A. Lowe 2 1 Department of Biochemistry, Molecular Biology, and Biophysics, Medical School, University of Minnesota, Minneapolis, Minnesota, United States of America, 2 Department of Physical Medicine and Rehabilitation, Medical School, University of Minnesota, Minneapolis, Minnesota, United States of America, 3 Division of Physical Therapy, Georgia State University, Atlanta, Georgia, United States of America Abstract Background: Estrogens are associated with the loss of skeletal muscle strength in women with age. Ovarian hormone removal by ovariectomy in mice leads to a loss of muscle strength, which is reversed with 17b-estradiol replacement. Aging is also associated with an increase in antioxidant stress, and estrogens can improve antioxidant status via their interaction with estrogen receptors (ER) to regulate antioxidant gene expression. The purpose of this study was to determine if ER and antioxidant gene expression in skeletal muscle are responsive to changes in circulating estradiol, and if ERs regulate antioxidant gene expression in this tissue. Methodology/Principal Findings: Adult C57BL/6 mice underwent ovariectomies or sham surgeries to remove circulating estrogens. These mice were implanted with placebo or 17b-estradiol pellets acutely or chronically. A separate experiment examined mice that received weekly injections of Faslodex to chronically block ERs. Skeletal muscles were analyzed for expression of ER genes and proteins and antioxidant genes. ERa was the most abundant, followed by Gper and ERb in both soleus and EDL muscles. The loss of estrogens through ovariectomy induced ERa gene and protein expression in the soleus, EDL, and TA muscles at both the acute and chronic time points. Gpx3 mRNA was also induced both acutely and chronically in all 3 muscles in mice receiving 17b-estradiol. When ERs were blocked using Faslodex, Gpx3 mRNA was downregulated in the soleus muscle, but not the EDL and TA muscles. Conclusions/Significance: These data suggest that Gpx3 and ERa gene expression are sensitive to circulating estrogens in skeletal muscle. ERs may regulate Gpx3 gene expression in the soleus muscle, but skeletal muscle regulation of Gpx3 via ERs is dependent upon muscle type. Further work is needed to determine the indirect effects of estrogen and ERa on Gpx3 expression in skeletal muscle, and their importance in the aging process. Citation: Baltgalvis KA, Greising SM, Warren GL, Lowe DA (2010) Estrogen Regulates Estrogen Receptors and Antioxidant Gene Expression in Mouse Skeletal Muscle. PLoS ONE 5(4): e10164. doi:10.1371/journal.pone.0010164 Editor: Maurizio C. Capogrossi, Istituto Dermopatico dell’Immacolata, Italy Received November 11, 2009; Accepted March 22, 2010; Published April 13, 2010 Copyright: ß 2010 Baltgalvis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a grant awarded to Dawn A. Lowe from the NIH (R01AG031743). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: baltg001@umn.edu Introduction There has been debate as to whether or not estrogen affects the force-generating capacity of skeletal muscle. A recent meta-analysis was conducted by our lab examining 23 studies in which postmenopausal women who were and were not taking estrogen hormone replacement were subjected to tests of muscle strength [1]. Our findings demonstrated a significant effect of approximately 5% greater strength in women who were taking estrogen replacement therapy. These findings are corroborated by a recent study that examined muscle strength in twins, in which one twin took estrogen replacement, and the other did not [2]. This paper showed that the hormone replacement therapy users walked at a maximal speed faster than non-users, and they had greater muscle power. We have extended the findings in humans using a mouse ovariectomy model. We showed that muscle and myosin functions were reduced ,20% in ovariectomized mice [3], and that those losses in force generation at both the whole muscle and molecular levels were completely restored when mice were administered 17b-estradiol [4]. How estradiol confers its beneficial effects to skeletal muscle and contractile proteins is not known. Theoretically, it could happen by non-genomic or genomic mechanisms. The most well-described mechanism for estradiol action in reproductive tissue is its genomic effects that are mediated through estrogen receptors (ER). In skeletal muscle, two isoforms of ERs have been identified, estrogen receptor a (ERa or Esr1) and estrogen receptor b (ERb or Esr2). These have been identified in multiple species including mice [5] and humans [6–9]. In human skeletal muscle, ERa mRNA levels are not different between males and females [6] and are expressed 180-fold greater than ERb mRNA [7]. More recently, ERa and ERb protein have been detected in human muscle, with about 2/3 of myonuclei staining positive for the receptors [7,9]. Less work has been done in mouse skeletal muscle. ERa mRNA was detected in mouse skeletal muscle, but ERb mRNA levels were undetectable [5]. In addition to ERa and ERb, a third isoform of the estrogen receptor, G-protein coupled receptor 30 (Gper or GPR30), has been recognized in several tissues, but is minimally expressed in skeletal muscle [10,11] and satellite cells [12]. PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e10164